Mixture of non-digestible oligosaccharides for stimulating the immune system

ABSTRACT

Nutritional compositions with fucosyllactose and betagalactooligosaccharides for use in stimulation of the immune system. The composition is suitable for infants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. patentapplication Ser. No. 13/383,823, filed Mar. 22, 2012, which is theNational Phase of International Patent Application No.PCT/NL2010/050446, filed Jul. 12, 2010, published on Jan. 20, 2011 as WO2011/008086 A1, which claims priority to U.S. Provisional ApplicationNo. 61/255,950, filed Oct. 29, 2009 and European Patent Application No.09165543.1, filed Jul. 15, 2009. The contents of these applications areherein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to nutrition with a mixture ofnon-digestible oligosaccharides, and in particular to the use thereoffor stimulating the immune system.

BACKGROUND OF THE INVENTION

Human milk fed infants have a lower incidence of infections, includingviral infections, than formula fed infants. Many components in humanmilk, including immunoglobulins (such as IgA), interleukin (IL)-1, IL-6,IL-8, IL-10, interferon-γ (IFN-γ), immunocompetent cells, transforminggrowth factor-β (TGF-β), lactoferrin, nucleotides, and non-digestibleoligosaccharides (NDO) are thought to be involved in protection againstinfection with enteric or respiratory pathogens.

NDO are a major constituent of human milk and are a major element of theinnate immune system of human milk. Human NDO promote the growth of abeneficial microbiota dominated by bifidobacteria and lactobacilli. Somehuman NDO are also known to be able to prevent directly the adhesion ofpathogens and toxins.

Human milk is the preferred food for infants. However, it is not alwayspossible or desirable to breast feed an infant. In such cases infantformulae or follow on formulae are a good alternative. These formulaeshould have an optimal composition in order to mimic the beneficialeffects of breast milk as close as possible.

WO 2007/067053 discloses infant formula comprising the plant-derivedprebiotics inulin and galacturonic acid oligosaccharide and the fromlactose synthesized prebiotic transgalacto-oligosaccharide to reduceinfections.

WO 2005/039597 relates to the use of acid oligosaccharide and neutraloligosaccharide for enhancing the immune system and the treatment and/orprevention of immune system related disorders.

WO 01/642255 relates to a nutritional composition comprising a prebioticfor enhancement of an immune response.

U.S. Pat. No. 6,576,251 discloses a carbohydrate mixture for dieteticfoods administered by the enteral or parenteral route consisting of (a)monosaccharide(s), (b) oligosaccharide(s) (at most hexasaccharides) and(c) polysaccharide(s) (at least heptasaccharides), where the mixingratio a, b, c, in respect of weight, is: alpha=1, b=40 to 1000, and c=1to 50, and containing at least 1 weight percent of fucose occurringeither freely and/or bound to an oligosaccharide and/or apolysaccharide. The carbohydrate mixture is said to have both anutritional and a biological effect which is considerably greater thanthe corresponding action of the individual constituents.

WO 99/11773 relates to methods of producing non-human transgenic mammalswhich produce various oligosaccharides and glycoconjugates in theirmilk. Subject matter that is claimed in this document relates to themammals themselves, the milk which they produce, compositions comprisingthe milk, fractions of the milk, and the purified oligosaccharides, aswell as glycoconjugates, present in the milk.

WO 2005/055944 discloses a pharmaceutical composition comprising amolecule comprising a fucose group in an α-1, 2 linkage, an α-1, 3linkage or an α-1, 4 linkage to a galactose group and a pharmaceuticallyacceptable carrier.

WO 2007/105945 relates to a food or supplement for pregnant womencomprising water soluble, non-digestible saccharides. The composition isused to improve the flora and/or immune system of the pregnant women, toimprove the immune system of the infant and to improve the intestinalflora of the infant after birth.

EP 1 629 850 provides a method and composition for the treatment and/orprevention of respiratory tract infection and/or respiratory tractinfection disease, said method comprising orally administering acomposition to a mammal, said composition comprising a galactosecontaining indigestible oligosaccharide and at least 5 wt. % digestiblegalactose saccharide.

Much effort is dedicated to find further solutions for balancing andstimulating the immune system.

SUMMARY OF THE INVENTION

Human milk differs from milk from domestic animals in that it comprisesmore NDO and that the NDO are structurally different. Human NDO is verycomplex, since it represents a heterogenic group of more than 130different compounds with diverse sugar composition. Because of theircomplex and polymorphic structure, large-scale synthesis is complicated.It is therefore not yet technically and economically feasible to prepareinfant nutrition with an NDO composition identical to human milk.

Recently, new techniques have become available to chemically synthesisespecific types of NDO identical to specific human NDO, thereby offeringthe opportunity to test the immunomodulatory capacity of specific humanNDO in in vitro and in vivo assays.

The inventors unexpectedly have found that fucosyllactose (FL), anoligosaccharide present in human breast milk and with a relativelysimple structure, when combined with betagalacto-oligosaccharides andpreferably also with fructo-oligosaccharides and/or uronic acidoligosaccharides, showed a synergistic effect on stimulating the immunesystem. An increased response in delayed type hypersensitivity reaction,indicative for an increased Th1 response, was observed after vaccinationwith an influenza vaccine in animals having consumed the presentcombination of FL and betagalacto-oligosaccharides, compared to animalshaving consumed the single components.

It was found that the present combination, i.e. the combinationcomprising fucosyllactose and betagalacto-oligosaccharides andpreferably further comprising fructo-oligosaccharides and/or uronic acidoligosaccharides increased the Th1 response. The effect observed wasunexpectedly higher than the sum of the effects of the single components

The present combination is especially advantageous for human subjectshaving a reduced Th1 response in comparison with healthy adults, inparticular newborn infants, elderly humans suffering fromimmunosenescence, humans suffering from AIDS or being infected with theHuman Immunodeficiency Virus, and cancer patients that are or have beensubjected to chemotherapy, radiation and cancer patients that arecachectic.

The present combination is suitable for treatment and/or prevention ofinfections, and/or for supporting vaccination response before, duringand/or after vaccination.

The present combination is especially suitable for the treatment and/orprevention of diseases which can be prevented and/or treated by anincrease in Th1 response and/or Th1/Th2 balance, in particular allergy,atopic dermatitis, asthma, food allergy, allergic rhinitis (e. g. pollenallergy), dust mite allergy and other forms of hypersensitivity likesystemic anaphylaxis and acute urticaria.

DETAILED DESCRIPTION

The present invention thus concerns a nutritional composition, not beinghuman milk, comprising fucosyllactose and betagalacto-oligosaccharides.

In the context of this invention the combination comprisingfucosyllactose and betagalacto-oligosaccharides and preferably furthercomprising fructo-oligosaccharides and/or uronic acid oligosaccharidesis also referred to as the present combination. In the context of thisinvention, the terms a composition accordin to the invention or thecomposition or thepresen tcomposition means a composition comprising thepresent combination.

Fucosyllactose

The present combination comprises fucosyllactose. Fucosyllactose (FL) isa non-digestible oligosaccharide present in human milk. It is notpresent in bovine milk. It consists of three monose units, fucose,galactose and glucose linked together. Lactose is a galactose unitlinked to a glucose unit via a beta 1,4 linkage. A fucose unit is linkedto a galactose unit of a lactose molecule via an alpha 1,2 linkage(2′-fucosyllactose, 2′-FL) or via an alpha 1,3 linkage to the glucoseunit of a lactose (3-Fucosyllactose, 3-FL). The present compositionpreferably comprises 2′-FL.

2′-FL, preferably α-L-Fuc-(1→2)-β-D-Gal-(1→4)-D-Glc, and 3-FL,preferably α-L-Fuc-(1→3)-[β-D-Gal-(1→4)]-D-Glc), are commerciallyavailable for instance from Sigma-Aldrich. Alternatively, they can beisolated from human milk, for example as described in Andersson &Donald, 1981, J Chromatogr. 211:170-1744, or produced by geneticallymodified micro-organisms, for example as described in Albermann et al,2001, Carbohydrate Res. 334:97-103.

Preferably, a composition according to the invention comprises 1 mg to 3g fucosyllactose per 100 ml, more preferably 10 mg to 2 g, even morepreferably 20 mg to 100 mg FL per 100 ml. Based on dry weight, thepresent composition preferably comprises 0.007 wt % to 20 wt %fucosyllactose, more preferably 0.07 wt % to 10 wt %, even morepreferably 0.15 wt % to 1 wt %. A lower amount of fucosyllactose will beless effective in stimulating the immune system, whereas a too highamount will result in unnecessary high costs of the product.

Betagalacto-oligosaccharides,

The present combination comprises betagalacto-oligosaccharides. It wasfound that the presence of betagalacto-oligosaccharides other than FLtogether with FL have a synergistic effect on immune stimulation, inparticular vaccination response.

Betagalacto-oligosaccharide can also be referred to astransgalacto-oligosaccharide. In a particularly preferred embodiment thepresent composition comprises betagalacto-oligosaccharides([galactose]n-glucose; wherein n is an integer from 2 to 60, i.e. 2, 3,4, 5, 6, . . . , 59 ,60; preferably n is selected from 2, 3, 4, 5, 6, 7,8, 9, or 10), wherein the galactose units are in majority linkedtogether via a beta linkage. Betagalacto-oligosaccharides (TOS) are forexample sold under the trademark Vivinal™ (Borculo Domo Ingredients,Netherlands). Another suitable source is Bi2Munno (Classado). Preferablythe betagalacto-ologosaccharides comprises beta 1,4 and beta 1,6linkages. Preferably the betagalacto-oligosaccharides have over 80% beta1,4 and beta 1,6 linkages, more preferably over 90% based on totallinkages linking the monomeric carbohydrate units.

Preferably the present composition comprises additional non-digestibleoligosaccharides with a DP between 2 and 250, more preferably 2 to 60.The non-digestible oligosaccharide is preferably at least one, morepreferably at least two, selected from the group consisting offructo-oligosaccharides and uronic acid oligosaccharides.

The group of fructo-oligosaccharides includes inulins.Fructo-oligosaccharide is a NDO comprising a chain of beta-linkedfructose units with a DP or average DP of 2 to 250, more preferably 2 to100, even more preferably 10 to 60. Fructo-oligosaccharide includesinulin, levan and/or a mixed type of polyfructan. An especiallypreferred fructo-oligosaccharide is inulin. Fructo-oligosaccharidesuitable for use in the compositions is also commercially available,e.g. Raftiline®HP (Orafti). Preferably the fructo-oligosaccharide has anaverage DP above 20.

The group of uronic acid oligosaccharides includes mannuronic acid,guluronic acid, galacturonic acid oligosaccharides, alginatedectradation products and pectin degradation products. Uronic acidoligosaccharides are preferably obtained from pectin degradationproducts. Hence the present composition preferably comprises a pectindegradation product with a DP between 2 and 100. Preferably the pectindegradation product is prepared from apple pectin, beet pectin and/orcitrus pectin. Preferably the uronic acid oligosaccharide is agalacturonic acid oligosaccharide.

In a preferred embodiment the composition comprises a mixture ofbetagalacto-oligosaccharides and fructo-oligosaccharides selected fromthe group consisting of short chain fructo-oligosaccharides and inulin,more preferably inulin. A mixture of at least two differentnon-digestible oligosaccharides advantageously stimulates the beneficialbacteria of the intestinal microbiota to a greater extent. Preferablythe weight ratio in a mixture of betagalacto-oligosaccharides andfructo-oligosaccharide, is between 25 and 0.05, more preferably between20 and 1. Preferably the present composition comprisesbetagalacto-oligosaccharides with a degree of polymerization (DP) of 2to 10 and/or fructo-oligosaccharides with a DP of 2 to 60.

Besides FL, most preferably the composition comprisesbetagalacto-oligosaccharide, fructo-oligosaccharide and a uronic acidoligosaccharide. It was found that such a combination actssynergistically with fucosyllactose, in particular 2′-fucosyllactose.The weight ratiobetagalacto-oligosaccharide:fructo-oligosaccharide:uronic acidoligosaccharide is preferably (20 to 2):1:(1 to 20), more preferably (20to 2):1:(1 to 10), even more preferably (20 to 2):1:(1 to 3), even morepreferably (12 to 7):1:(1 to 2). Most preferably the weight ratio isabout 9:1:1.1. Preferably the weight ratio FL tobetagalacto-oligosaccharide, preferably TOS, is from 5 to 0.05, morepreferably 5 to 0.1, more preferably from 2 to 0.1. Preferably theweight ratio FL to fructo-oligosaccharide, preferably inulin, is from 10to 0.05, more preferably 10 to 0.1, more preferably from 2 to 0.5.Preferably the weight ratio FL to uronic acid oligosaccharide,preferably derived from pectin, is from 10 to 0.05, more preferably 10to 0.1 more preferably from 2 to 0.5.

Preferably, the composition comprises 80 mg to 4 g non-digestibleoligosaccharides, including fucosyllactose andbetagalacto-oligosaccharides, per 100 ml, more preferably 150 mg to 2 g,even more preferably 300 mg to 1 g non-digestible oligosaccharides per100 ml. Based on dry weight, the composition preferably comprises 0.25wt % to 25 wt % non-digestible oligosaccharides, more preferably 0.5 wt% to 10 wt %, even more preferably 1.5 wt % to 7.5 wt %. A lower amountof non-digestible oligosaccharides will be less effective in stimulatingthe beneficial bacteria in the microbiota, whereas a too high amountwill result in side-effects of bloating and abdominal discomfort.

Nutritional Composition

The present combination of FL and betagalato-oligosaccharides ispreferably a nutritional composition. The composition of the presentinvention is not human milk. The present composition is preferablyenterally administered, more preferably orally.

The present composition is preferably a nutritional formula, preferablyan infant formula. The present composition can be advantageously appliedas a complete nutrition for infants. The present composition preferablycomprises a lipid component, protein component and carbohydratecomponent and is preferably administered in liquid form. The presentinvention includes dry food, preferably a. powders which is accompaniedwith instructions as to admix said dry food mixture with a suitableliquid, preferably with. water.

The present invention advantageously provides a composition wherein thelipid component provides 5 to 50% of the total calories, the proteincomponent provides 5 to 50% of the total calories, and the digestiblecarbohydrate component provides 15 to 85% of the total calories. Thepresent invention advantageously provides a composition wherein thelipid component provides 20 to 50% of the total calories, the proteincomponent provides 5 to 30% of the total calories, and the digestiblecarbohydrate component provides 30 to 70% of the total calories.Preferably, in the present composition the lipid component provides 35to 50% of the total calories, the protein component provides 7.5 to12.5% of the total calories, and the digestible carbohydrate componentprovides 40 to 55% of the total calories. For calculation of the % oftotal calories for the protein component, the total of energy providedby the proteins, peptides and amino acids needs to be taken intoaccount.

The present composition preferably comprises at least one lipid selectedfrom the group consisting of animal lipid, excluding human lipids, andvegetable lipids. Preferably the present composition comprises acombination of vegetable lipids and at least one oil selected from thegroup consisting of fish oil, animal oil, algae oil, fungal oil, andbacterial oil. The present composition preferably comprises long chainpoly-unsaturated fatty acids (LC-PUFA). LC-PUFA are fatty acids or fattyacyl chains with a length of 20 to 24 carbon atoms, preferably 20 or 22carbon atoms comprising two or more unsaturated bonds. More preferablythe present composition comprises eicosapentaenoic acid (EPA, n-3),docosahexaenoic acid (DHA, n-3) and/or arachidonic acid (ARA, n-6).

Preferably the present composition comprises at least 0.1 wt. %,preferably at least 0.25 wt. %, more preferably at least 0.6 wt. %, evenmore preferably at least 0.75 wt. % LC-PUFA with 20 and 22 carbon atomsbased on total fat content.

The content of LC-PUFA, particularly the LC-PUFA with 20 and 22 carbonatoms, preferably does not exceed 6 wt %, more preferably does notexceed 3 wt. % of the total fat content as it is desirable to mimichuman milk as closely as possible. The LC-PUFA may be provided as freefatty acids, in triglyceride form, in diglyceride form, in monoglycerideform, in phospholipid form, or as a mixture of one of more of the above.The present composition preferably comprises between 5 and 75 wt. %polyunsaturated fatty acids based on total fat, preferably between 10and 50 wt. %.

The protein used in the nutritional composition is preferably selectedfrom the group consisting of non-human animal proteins (preferably milkproteins), vegetable proteins (preferably soy protein and/or riceprotein), hydrolysates thereof, free amino acids and mixtures thereof.The present composition preferably contains casein, whey, hydrolyzedcasein and/or hydrolyzed whey protein. Preferably the protein comprisesintact proteins, more preferably intact bovine whey proteins and/orintact bovine casein proteins.

The present composition preferably contains digestible carbohydratesselected from the group consisting of sucrose, lactose, glucose,fructose, corn syrup solids, starch and maltodextrins, more preferablylactose.

In view of the above, it is also important that the liquid food does nothave an excessive caloric density, however still provides sufficientcalories to feed the subject. Hence, the liquid food preferably has acaloric density between 0.1 and 2.5 kcal/ml, even more preferably acaloric density of between 0.5 and 1.5 kcal/ml, most preferably between0.6 and 0.8 kcal/ml.

Preferably the present composition comprises nucleotides and/ornucleosides, more preferably nucleotides. Preferably, the compositioncomprises cytidine 5′-monophospate, uridine 5′-monophospate, adenosine5′-monophospate, guanosine 5′-monophospate, and/or inosine5′-monophospate, more preferably cytidine 5′-monophospate, uridine5′-monophospate, adenosine 5′-monophospate, guanosine 5′-monophospate,and inosine 5′-monophospate. Preferably the composition comprises 5 to100, more preferably 5 to 50 mg, most preferably 10 to 50 mg nucleotidesand/or nucleosides per 100 gram dry weight of the composition. Thepresence of nucleotides and/or nucleotides advantageously stimulate NKcell activity. The nucleotides and/or nucleosides are deemed to actsynergistically with the fucosyllactose of the present composition.

Application

The present combination of FL and betagalato-oligosaccharides was foundto synergistically stimulate the immune-system. In particularly the Th1response was increased. The effect of the combination of these twocomponents is higher than the sum of the effects of the singlecomponents.

The present combination can advantageously be used in the treatmentand/or prevention of a disease, and thus the invention concerns a methodfor the treatment and/or prevention of a disease in a mammal, saidmethod comprising administering the present combination to the mammal.In other words, the invention also concerns the use of a combinationaccording to the present invention for the manufacture of a composition,preferably a nutritional composition, for the treatment and/orprevention of a disease. In other words the invention concerns acomposition or nutritional composition comprising a combinationaccording to the present invention for use in the treatment and/orprevention of a disease. Preferably the mammal is a human, even morepreferably a human infant. Thus the invention also concerns the use of acombination according to the present invention for the manufacture of acomposition, preferably a nutritional preparation, for the treatmentand/or prevention of a disease in an infant. Or in other words theinvention concerns a composition or nutritional composition comprising acombination according to the present invention for use in the treatmentand/or prevention of a disease in an infant.

In the context of this invention, an infant is in the age of 0 to 6years, preferably in the age of 0 to 4 years, preferably in the age of 0to 2 years, preferably in the age of 0 to 1 year.

Also the invention concerns a method for providing nutrition to aninfant, said method comprising administering the present combination ornutitional composition to the infant. In other words, the invention alsoconcerns the use of a combination according to the present invention forthe manufacture of a nutritional composition for providing nutrition toan infant. In other words the invention concerns a composition ornutritional composition comprisng a combination according to the presentinvention for use in providing nutrition to an infant.

The present combination can advantageously be used to increase the Th1response, increase the Th1/Th2 balance, restore imbalance in the Th1/Th2responses, maintain a favorable Th1/Th2 balance and/or for the treatmentand prevention of disorders which are associated with a Th1/Th2imbalance. Hence, compositions which are advertised to e.g. simulatematuration of the immune system, enhance the resistance to pathogens byenhancing the immune system and/or support the immune system are part ofthe present invention. In a further aspect, the present inventionprovides a method for the treatment and/or prevention of an immunesystem related disorder, said method comprising administering to saidmammal a composition comprising a therapeutically effective amount ofthe present combination. In a further aspect, the present inventionprovides a method of enhancing the immune response in a mammal saidmethod comprising administering to the mammal the present combination.

The immune system of newborn human infants is characterized by an excessof Th2 response. During maturation of the immune system, the Th1response increases and the Th1/Th2 balance shifts to values observed forhealthy adults. Hence, the present combination is especiallyadvantageous for human infants. The present invention supports thematuration of the immune system in infants. In a further embodiment, themethod of the invention relates to the administration of the presentcombination to humans in the age of 0 to 6 years, preferably in the ageof 0 to 4 years, preferably in the age of 0 to 2 years, more preferablyin the age of 0 to 1 year. In a preferred embodiment the present methodrelates to the stimulation of the maturation of the immune system inhuman subjects in the age of 0-6 years, preferably in the age of 0 to 4years, preferably in the age of 0 to 2 years, more preferably in the ageof 0 to 1 year.

The composition comprising fucosyllactose andbetagalacto-oligosaccharides even more advantageously is used in preterminfants and/or very low or low birth weight infants, since these infantsare even more vulnerable and/or prone to viral infections.

The composition comprising fucosyllactose andbetagalacto-oligosaccharide even more advantageously is used in infantsdelivered via Caesarean section. Caesarean section born infants are bornin a hospital in an environment having more pathogens against which theantibodies, conferred by the mother to the infant, are not effective.Caesarean section born infants have a delayed and less optimalcolonization of the large intestinal tract and therefore are also moreprone to intestinal infections

A too low Th1/Th2 balance leads to extreme sensitivity towards foreigncomponents which could lead to a variety of immunological reactions,e.g. allergies and related diseases such as atopic dermatitis, asthma,food allergy, allergic rhinitis (e. g. pollen allergy), dust miteallergy and other forms of hypersensitivity like systemic anaphylaxisand acute urticaria. Hence, the present combination is especiallyadvantageous for treatment and/or prevention of a disorder selected fromthe group consisting of allergy, food allergy, atopic dermatitis,asthma, allergic rhinitis, dust mite allergy and urticaria. The presentinvention increases the Th1/Th2 balance.

An increase in Th1 response leads to an increase in the response againstpathogenic bacteria and/or viruses. Hence, the present combination issuitable for the treatment and/or prevention of infections. The presentpreparation can be advantageously used for the treatment and/orprevention of intestinal infections, systemic infections and/orrespiratory tract infections.

Since the combination of FL and betagalacto-oligosaccharides was foundto specificially synergistically enhance the Th1 response, the presentinvention is particularly suitable to prevent viral infections, morepreferably viral infections caused by orthomyxoviridae, in particularthe influenza virus, herpesviridae, rotavirus, cytomegalovirus,caliciviridae, respiratory syncytial virus, human imunofeficiency virusand/or rhinovirus. The use of the present invention is thereforepreferably for preventing and/or treating viral infections, morepreferably the viral infections common cold, flu, measles, chicken pox,viral diarrhoea, viral gastro-enteritis, HIV infection and/or viralrespiratory tract infections.

It was also found that the present combination can suitably be used tosupport vaccination processes, e.g. enhance the effects of a vaccinationprocess. The present combination is suitable for supporting vaccinationresponse before, during and/or after vaccination. Particularly theeffects of vaccinations for diptheria-tetanus, pertussis, polio vaccine,measles/mumps/rubella, pneumococcal conjugate, haemophilus B conjugate,hepatitis B, hepatitis A, varicella, andor influenza can suitably beenhanced. Hence, the present combination is advantageously used in thetreatment and/or prevention of infections, and/or for use in enhancementof vaccination response.

Hence, the present combination is advantageous for human subjectssuffering from immune deficiencies, in particular elderly humanssuffering from immunosenescence, humans suffering from AIDS or beinginfected with the Human Immunodeficiency Virus, and/or cancer patients,more particular cancer patients that are or have been subjected tochemotherapy, radiation and cancer patients that are cachectic.

The present composition is advantageously used for nutriton for elderly.Elderly have a decreased Th1 response. Elderly are especially vulnerableto viral infection complications. In a preferred embodiment the presentinvention is used for treatment and/or prevention of immunosenescence inelderly. In one embodiment, the present invention concerns providingnutrition to an elderly person. An elderly person is a person having anage of 55 years or more, in particular of the age of 65 or more.

EXAMPLES Example 1

The effect of diets comprising 2′-FL and/or betagalacto-oligosaccharideswas tested in a mouse model wherein a response to an antigen is measuredby a delayed-type hypersensitivity (DTH) response. This DTH response inthe ears after local challenge with an antigen present in a vaccine is ameasure of Th1 cell proliferation. During response to infection and/orvaccination Th1 cells proliferate in response to the challenge with theantigen. These Th1 cells infiltrate the ear when the ear is subsequentlychallenged with the antigen and cause swelling. Infiltration with theTh1 cells in the ear takes about 24 h and the swelling is thereforedelayed. The more Th1 cells proliferated during initial vaccinationand/or infection, the more DTH upon challenging with the antigen isobserved.

Materials and Methods

6-8 Weeks old female C57BL/6 mice (Charles River) received semi-purifiedAIN-93G-based diets (Research Diet Service, Wijk bij Duurstede, theNetherlands), comprising 1) 1 wt % betagalacto-oligosaccharide (GOS,source Vivinal GOS, Borculo Domo), fructo-oligosaccharide (FOS, SourceRaftilineHP, Orafti) and galacturonic acid oligosaccharide (source AOS)in a 9:1:1.1 ratio. AOS were produced from pectin (Südzucker A G,Mannheim, Germany), with a DP of 1-20. It consists of approximately 75%galacturonic acid oligomers, based on total weight,

2) 1 wt % 2′fucosyllactose (2′-FL),

3) 1 wt % betagalacto-oligosaccharides, fructo-oligosaccharides andgalacturonic acid oligosaccharide (AOS) in a 9:1:1.1 ratio and 1 wt %2′-FL.

4) 0.5 wt % betagalacto-oligosaccharides, fructo-oligosaccharides andgalacturonic acid oligosaccharide (AOS) in a 9:1:1.1 ratio and 0.5 wt %2′-FL.

All groups were compared to the unsupplemented control diet (control andsham control group). Dietary supplementation started 14 days before thefirst vaccination and lasted until the end of the experiment, 31 daysafter the first vaccination.

Vaccination experiments were performed using Influvac (SolvayPharmaceuticals, Weesp, the Netherlands) from season 2005/2006. The micereceived a primary vaccination and a booster vaccination, consisting ofa subcutaneous (sc) injection of a 1:1 mix of vaccine and adjuvant in atotal volume of 100 μl. The booster vaccination was given at 21 daysafter the primary vaccination. The experiments ended 10 days afterbooster vaccination. Negative control groups that were included receivedinjections with a 1:1 mix of PBS and adjuvant in a total volume of 100μl (sham control). Ear thickness was measured in duplicate beforevaccine challenge and 24 h thereafter, using a digital micrometer(Mitutoyo Digimatic 293561, Veenendaal, the Netherlands). The DTHresponse was calculated by subtracting the basal ear thickness from thevalue at 24 h after challenge.

Results

Supplementation with GOS/FOS/AOS, or 2′ FL alone resulted in anincreased ear swelling. However, the combination of 2′FL and GOS/FOS/AOSresulted in an even higher increase of the DTH response, a TH1-dependentparameter, compared with control-fed animals and compared with thesingle components. The combination synergistically increased vaccinationresponse. See Table 1.

TABLE 1 Effect of 2′-FL and TOS on DTH response. Earswelling Dietaryintervention DTH (se) Δ DTH μm Relative DTH Sham control 19.3 (5.5) 0 0Control 66.3 (6.4) 47.0 1 1% TOS/FOS/AOS 90.8 (7.5) 71.5 1.52 1% 2′-FL99.7* (10.2) 80.4 1.71 0.5% TOS/FOS/AOS, 117.8** (4.4)   98.5 2.10 0.5%2′-FL expected 95.3 76.0 1.62 *indicates p < 0.05 compared to controlgroup **indicates p < 0.01 compared to control group and p < 0.05compared to 0.5% GOS/FOS/AOS.

As the effect on DTH response is significantly higher (110%) than theDTH responses from diets containing the FL (71%) orbetagalacto-oligosaccharides (52%) alone, and also much higher thanbased on the additive effect of which an be calculated to be 62%increase of DTH, these results are indicative for the synergistic effectprovided by the administration of FL and betagalato-oligosaccharides,and preferably fructo-oligosaccharides and/or uronic acidoligosaccharides, on Th1 response increase.

Overall, these results support that oral supplementation with FL incombination with betagalacto-oligosaccharides, more preferablyadditionaly comprising fructo-oligosaccharides and/or uronic acidoligosaccharides, stimulates the immune response. The immune response isin particular enhanced with respect to the Th1 response and/or thevaccination response. The results are also indicative for an increasedTh1/Th2 balance.

The results of this experiment are an indication that the presentinvention can advantageously be used for support in vaccinationresponse. The results of this experiment are also an indication that itcan advantageously be used in subjects with a low Th1 response, inparticular infants. The results of this experiment are also anindication that it can advantageously be used in subjects with a low Th1response, in particular elderly suffering or at risk for suffering fromimmunosenescence, HIV patients, AIDS patients and/or cancer patientsthat are or have been subjected to chemotherapy and/or radiation or thatare cachectic. This model is indicative for basic immunological changes,which can be beneficial in all disorders with malfunctioning immunesystem.

Example 2

Infant formula for stimulating immune system comprising per 100 ml (13.9dry weight):

1.4 g protein (whey and casein)

7.3 g digestible carbohydrates (including lactose)

3.6 g fat (vegetable fat, fish oil)

0.8 g non-digestible oligosaccharides of which 80 mg 2′-fucosyllactoseand 640 mg beta-galacto-oligosaccharides, and 80 mgfructo-oligosaccharides

Further are included: choline, myo-inositol, taurine, minerals, traceelements, and vitamins as known in the art.

Example 3

Infant formula for stimulating immune system comprising per 100 ml (13.9dry weight):

1.4 g protein (whey and casein)

7.3 g digestible carbohydrates (including lactose)

3.6 g fat (vegetable fat, fish oil)

0.8 g non-digestible oligosaccharides of which 40 mg 2′-fucosyllactoseand 760 mg beta-galacto-oligosaccharides, fructo-oligosaccharides anduronic acid oligosaccharides in a 9:1:1.2 wt/wt ratio.

Further are included: choline, myo-inositol, taurine, minerals, traceelements, and vitamins as known in the art.

1-14. (canceled)
 15. A nutritional composition, not being human milk,comprising fucosyllactose and betagalacto-oligosaccharides.
 16. Thenutritional composition according to claim 15, further comprisingfructo-oligosaccharides and/or uronic acid oligosaccharides.
 17. Thenutritional composition according to claim 15, further comprisingfructo-oligosaccharides and uronic acid oligosaccharides.
 18. Thenutritional composition according to claim 15, wherein fucosyllactose is2′-fucosyllactose.
 19. The nutritional composition according to claim15, wherein the betagalacto-oligosaccharides have over 80% beta 1,4 andbeta 1,6 linkages.
 20. The nutritional composition according to claim 15comprising protein, digestible carbohydrates and fat, wherein proteinprovides 5 to 50%, digestible carbohydrates provide 15 to 85% , and fatprovides 5 to 50% of calories based on total calories.
 21. Thenutritional composition according to claim 15, having 0.07 to 1 wt.%2′-fucosyllactose and/or 0.25 wt % to 15 wt % of the sum ofbetagalacto-oligosaccharides, fructo-oligosaccharides and uronic acidoligosaccharides based on dry weight of the composition.
 22. A method ofstimulating the immune system, comprising administering to a mammal inneed thereof a composition comprising fucosyllactose andbetagalacto-oligosaccharides said composition not being human milk. 23.The method according to claim 22, wherein the immune system isstimulated by increasing Th1 response, or increasing Th1/Th2 balance orboth.
 24. The method according to claim 22, wherein the mammal is aninfant.
 25. A method of enhancement of a vaccination response in amammal, comprising administering to the mammal a composition comprisingfucosyllactose and betagalacto-oligosaccharides said composition notbeing human milk.
 26. The method according to claim 25, wherein themammal is an infant.
 27. A method of treatment and/or prevention ofinfections, comprising administering to a mammal in need thereof acomposition comprising fucosyllactose and betagalacto-oligosaccharidessaid composition not being human milk.
 28. The method according to claim27, wherein the mammal is an infant.